The long-range goal of our work is to understand the function and significance of tropomyosin (TM) isoform diversity in nonmuscle cells and the relationship of TM expression to cell growth and the transformed phenotype. Although fibroblasts are known to express at least six isoforms of TM, the functions of the different isoforms are not known. In vitro, TM isoforms demonstrate differences in affinity and co- operativity of binding to actin filaments. The relationship of these differences to the regulation of actin filament assembly and the binding of other microfilament associated proteins is not fully understood. In vivo, TMs are generally localized to stabilized assemblies of micro- filament bundles, such as stress fibers. Studies have demonstrated profound alterations in the pattern of tropomyosin isoform expression in cells transformed by oncogenic tumor viruses, UV irradiation, and chemical carcinogens. This may in part be responsible for the reduction of stress fibers and the accompanying alterations of cell shape characteristic of transformed cells. Although intriguing, a causal relationship between the changes in cytoarchitecture and cell shape and the altered pattern of TM isoform expression in transformed cells has yet to be demonstrated. The experiments outlined in this grant will use a combination of in vitro and in vivo approaches to study the function of TM diversity. Homogeneous preparations of each fibroblast isoform will be prepared using bacteria and insect cell expression systems. These proteins will be used to study the biochemical properties of the different isoforms such as actin binding, head-to-tail interactions, effects on actin filament depolymerization, and interactions of TM with other micro- filament associated proteins such as caldesmon and gelsolin. Individual TM isoforms will be fluorescently labeled and microinjected into living mammalian cells. The dynamic distribution of these proteins will be analyzed using a fluorescent microscope equipped with a silicon- intensifier target camera and a time-lapse video recorder. The dynamic distribution of each isoform during cell respreading, mitosis, and migration will be determined. To complement these studies, isoform- specific antibodies will be prepared against synthetic peptides. The effects of altering the expression of specific TM isoforms in normal and transformed cells will be studied.